P743: THE PIVOTAL ROLE OF GLUTAMINOLYSIS IN MYELODYSPLASTIC SYNDROME (MDS): A NOVEL STRATEGY FOR THE TARGETED THERAPY OF MDS
Authors: S. Okabe, Y. Tanaka, A. Gotoh
Journal: HemaSphere (2022)

Background: Myelodysplastic syndromes (MDS) is indicated by bone marrow failure and increased risk of transformation to acute myelogenous leukemia (AML). MDS is more common with advancing age and a combination of comorbidities is associated with significantly worse overall survival. Moreover, iron overload (IOL) starts to develop in MDS patients by transfusion in many cases. Because the outcome of MDS is poor, alternative therapeutic strategies are required to improve the survival of MDS and AML patients. Aims: Glutamine is the most abundant amino acid in blood. Glutaminase (GLS) is the initial enzyme in glutamine metabolism and glutaminolysis contributes to tumor growth. Because GLS is frequently activated in various types of cancer, GLS inhibitor could suppress MDS and AML cells in combination with BCL2 inhibitor. Methods: In this study, we investigated whether GLS was involved in MDS progress. We also investigated the efficacy of GLS inhibitor (CB-839 or IPN-60090) and BCL2 inhibitor, venetoclax by using MDS and AML cell line, SKM-1, MDS-L, MOLM-14, THP-1, MV4;11 and osteoblastic cell line, MC3T3-E1. Results: We first investigated the relationship between GLS expression and MDS patients by microarray gene expression data from the online Gene Expression Omnibus (GEO). In mammalian cells, there are two paralogous GLS genes, GLS1 and GLS2. GLS1 expression was increased in refractory anemia with excess of blasts (RAEB)-2 cells compared to normal control cells (GSE19429). In contrast, GLS2 expression was not changed. High GLS1 expression is associated with poor prognosis in AML patients. Deprivation of glutamine in culture medium revealed that cellular growth inhibition. We next evaluated the effect of GLS inhibitor (CB-839 or IPN-60090) or venetoclax on proliferation of MDS and AML cell lines. 72 h treatment of MDS and AML cells were inhibited by GLS inhibitor or venetoclax in a dose dependent manner. Cellular cytotoxicity and caspase 3/7 activity was also increased. Glutamine is converted by GLS into glutamate and related nicotinamide adenine dinucleotide phosphate (NADP) production. Intracellular NADPH and NADH were reduced by GLS inhibitor. Co-treatment with GLS inhibitor and venetoclax was superior effect than single drug alone. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased. The bone marrow is a relatively hypoxic microenvironment. Gene expression of GLS1 is increased under hypoxia condition. The proteasome 20S activity and phosphorylation of nuclear factor-kappa B (NF-κB) were increased. Efficacy of venetoclax is reduced under hypoxia condition. Co-treatment with GLS inhibitor and venetoclax overcome in hypoxia mediated drug resistance. GLS1 shRNA transfected cells reduced cellular proliferation. Cell cycle analysis showed G1 arrest and increased sensitivity of venetoclax. Because MDS patients with IOL have reduced overall survival and poorer outcomes, we next examined IOL by using osteoblastic cell line, MC3T3-E1. The cellular proliferation was reduced by ferric ammonium citrate (FAC). Intracellular ROS was increased by FAC. GLS inhibitor treatment protected FAC mediated cell death. Summary/Conclusion: Targeting of glutaminolysis and BCL2 inhibition combine to enhance therapeutic efficacy and has been proposed as a novel strategy high-risk MDS and AML. We also provide the promising clinical relevance as a candidate drug for treatment of MDS and AML patients.